Biomedical subjects

V Vachon

Publications and source records attributed to V Vachon.

32 records · Page 2Linked to original sources

PubMed1995

Factors affecting the stability of the renal sodium/phosphate symporter during its solubilization and reconstitution.

Phosphate is reabsorbed across the brush-border membrane of the proximal tubule by a specific sodium-dependent symporter. Like the other brush-border membrane transport proteins of the kidney, the phosphate carrier remains to be isolated in a functional state. To establish a set of parameters that allow to preserve its biological activity, the phosphate carrier was solubilized under systematically varied conditions and reconstituted into proteoliposomes. Successful reconstitution was achieved only when the extraction buffer contained lipids extracted from the renal brush-border membrane. Glycerol, an osmolyte which reduces the water activity of the solution, was also required. It could however be replaced by 150 mM sodium or potassium phosphate. Below this concentration and in the presence of glycerol, the ionic strength of the solution had little effect on the stability of the transporter, but sodium phosphate could not be replaced by sodium chloride. Phosphate transport in reconstituted vesicles depended on the concentration of detergent and pH of the extraction buffer. Finally, transport activity was increased when solubilization was carried out in the presence of a reducing agent, dithiothreitol. These results should be helpful during the purification and further characterization of the renal phosphate symporter.

Animals↗

PubMed1994

Molecular size of the functional complex and protein subunits of the renal phosphate symporter.

The oligomeric structure of the rabbit renal brush-border membrane sodium/phosphate cotransporter was examined with the radiation inactivation and fragmentation technique. The size of its functional complex (its "radiation inactivation size") was estimated from the rate of decay of its sodium-dependent transport activity as a function of the radiation dose. A radiation inactivation size of 223 +/- 42 kDa was obtained. The polypeptide constituting the monomeric unit of the Na1+/Pi symporter was detected by immunoblotting with polyclonal anti-peptide antibodies directed against the 14 amino acid C-terminal portion of the symporter molecule. Its apparent molecular size estimated by comparison with standards following SDS-polyacrylamide gel electrophoresis was 64,000. This value is in good agreement with its known molecular mass of 51,797 Da calculated from the amino acid sequence deducted from the nucleotide sequence of its gene since this protein is probably glycosylated. The loss of labeling intensity of the polypeptide of M(r) = 64,000 was also measured as a function of radiation dose. The molecular size calculated from these data (its "target size") was 165 +/- 20 kDa. The target size estimated for the rat phosphate cotransporter was 184 +/- 46 kDa, and its previously reported radiation inactivation size was 234 +/- 14 kDa. These results strongly suggest that the renal Na1+/Pi cotransporter exists as an oligomeric protein, probably a homotetramer. The fact that the values obtained for the target size are about 3/4 those obtained for the radiation inactivation size of these cotransport proteins indicates that their subunits are closely associated since most of their subunits appear to be fragmented by a single ionizing radiation hit.

Animals↗

PubMed1994

Immunodetection and characterization of proteins implicated in renal sodium/phosphate cotransport.

Polyclonal antibodies raised against the 14-amino acid C-terminal portion of the rabbit renal brush-border membrane Na+/Pi cotransporter, as deduced from the nucleotide sequence of the cloned NaPi-1 gene, were used for Western blot analysis of renal brush-border membrane proteins from rat, rabbit and beef. Proteins of 65 kDa from the rat, 64 kDa from the rabbit, and 38, 66, 77, 92, 110, 176 and 222 kDa from the beef were specifically labelled. The affinity of the antibodies was much greater, however, for the proteins of the rat and rabbit than for those of the beef. The rat 65-kDa antigen was readily detected in brush-border membranes isolated from kidney cortex, but was absent from the basolateral membrane and the cytosolic and microsomal fractions of this tissue, in agreement with the subcellular localization of the Na+/Pi cotransporter. This antigen was however several-fold more abundant in the juxtamedullary portion of the cortex than in the outer portion. Despite a strong stimulation in phosphate transport, a low-phosphate diet had little influence on the amount of antigen detected. An additional peptide-displaceable band corresponding to a protein of 250 kDa appeared when beta-mercaptoethanol was omitted during electrophoresis, in agreement with the possibility that disulfide bonds may be involved in the regulation of renal phosphate transport activity.

Amino Acid Sequence↗

PubMed1994

Voltage gating of porins from Haemophilus influenzae type b.

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (M(r) 37,782; 341 amino acids). Porins were purified from Hib strains representative of the three outer membrane protein subtypes 1H, 2L and 6U, reconstituted into artificial planar bilayers, and tested for their voltage dependency. At membrane potentials of 50-80 mV, individual Hib 2L and 6U porin channels showed a high probability of undergoing a reversible change to one of several lower conducting substates. Such behaviour was not observed for Hib 1H porin with transmembrane potentials up to 80 mV. The voltage dependence of Hib 2L and 6U porins was asymmetric: it occurred at only one polarity. The asymmetry was also observed for membranes with numerous porins incorporated, suggesting that Hib porin inserted asymmetrically into the bilayer. At macroscopic levels the voltage gating reduced the conductance by 25-50%, implying that the channels closed only partially. Hib 2L porin differs from Hib 1H porin by the substitution Arg166Gln and Hib 6U porin differs from Hib 1H porin by substitutions at ten amino acids including the change Arg166Leu. We conclude that substitutions at Arg166 residue, which is localized to surface-exposed loop number four, are associated with a lowered threshold potential for the voltage gating of Hib porin. This surface-exposed loop may play some role in the conformational changes that occur during voltage gating.

Haemophilus influenzae↗

PubMed1992

Characterization of essential arginine residues implicated in the renal transport of phosphate and glucose.

We have characterized the reaction of arginine-specific reagents with the phosphate and glucose carriers of the kidney brush-border membrane. The inhibition of phosphate and glucose transport by phenylglyoxal follows pseudo-first-order kinetics. The rate of inactivation of phosphate transport by 50 mM phenylglyoxal was about 3-fold higher than that for glucose transport (kapp was 0.052 s-1 for the uptake of phosphate and 0.019 s-1 for the uptake of glucose). The order of the reaction, n, with respect to phenylglyoxal was 1.25 and 1.31 for the inactivation of phosphate and glucose transport, respectively. The inactivation of phosphate flux by p-hydroxyphenylglyoxal also follows pseudo-first-order kinetics, but the inhibition rate (kapp = 0.0012 s-1) was slower than with phenylglyoxal. The inactivation increased with the alkalinity of the preincubation medium for both phosphate and glucose fluxes and was maximal at pH 9.0. The inactivation of phosphate flux by phenylglyoxal depends upon the presence of an alkaline intravesicular pH. Extravesicular pH does not affect the reaction. Phenylglyoxal does not interfere with the recycling of the protonated carrier since phosphate uptake is inhibited independently of the pH used for transport measurements. Moreover, phenylglyoxal completely abolished trans stimulation by phosphate. Trans sodium inhibited phosphate uptake and abolished the pH profile of phosphate uptake.

Animals↗

PubMed1992

Molecular size of the renal sodium/phosphate symporter in native and reconstituted systems.

The size of the renal sodium/phosphate symporter was estimated with the radiation inactivation technique in isolated bovine brush border membrane vesicles and after reconstitution in proteoliposomes. The functional unit of the native phosphate carrier had a radiation inactivation size of 172 +/- 17 kDa. Identical values were obtained for the reconstituted carrier whether it was irradiated before or after the formation of the proteoliposomes (161 +/- 9 and 159 +/- 11 kDa, respectively). The sodium-independent uptake of phosphate was not affected significantly by radiation doses up to 10 Mrad. This activity is therefore not due to the reconstitution of a large phosphate-binding protein such as alkaline phosphatase. Furthermore, bromotetramisole, a specific inhibitor of phosphate binding to this enzyme, had no significant effect on the uptake of phosphate by the proteoliposomes.

Animals↗

PubMed1992

Recombinant porin of Haemophilus influenzae type b.

A protein of approximately 40 kDa in the outer membrane of Haemophilus influenzae type b (Hib) behaves as a porin and permits transmembrane diffusion of low-molecular-weight solutes. The gene for Hib porin was cloned from an M13 library of chromosomal DNA of Hib strain ATCC9795. The gene was subcloned into a new transfer vector as a prerequisite for its use in the baculovirus expression vector system. Pure recombinant virus (AcPORIN) was isolated. On infection of a cultivated insect cell line Sf9 with AcPORIN, a novel protein was detected in cell lysates, and this novel protein reacted with an anti-Hib porin monoclonal antibody. The purified recombinant Hib porin was tested for its pore-forming properties in a synthetic black lipid membrane. The biophysical activity of purified recombinant Hib porin was identical to porin isolated from the bacterial outer membrane.

Bacterial Outer Membrane Proteins↗

PubMed1992

Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities.

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38,000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial cell surface, seven mouse monoclonal anti-Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104-139; domain (ii) amino acids 162-174; and domain (iii), amino acids 267-341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement-mediated lysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface-binding, they were neither bactericidal nor protective.

Amino Acid Sequence↗

PubMed1991

Reconstitution of the renal brush-border membrane sodium/phosphate co-transporter.

A simple and rapid procedure was developed for the reconstitution of Na(+)-dependent phosphate-transport activity from bovine kidney brush-border membranes. The phosphate transporter appears to be particularly sensitive to extraction conditions. To prevent its inactivation, the phosphate carrier was solubilized in a buffer containing its substrates, Na+ and phosphate, CHAPS, dithiothreitol, brush-border membrane lipids and glycerol. The uptake of phosphate by reconstituted vesicles was strongly stimulated by the presence of a transmembrane Na+ gradient. This stimulation was abolished when the Na+ gradient was dissipated by monensin. The affinity of the carrier for phosphate was similar in proteoliposomes and in brush-border membrane vesicles (apparent Kt = 40 microM). The transporter was also stimulated by the presence of a high concentration of phosphate on the trans side of the membrane. The reconstituted transport activity was inhibited by arsenate, a known inhibitor of phosphate transport. However, the bovine phosphate carrier, intact or reconstituted, was much less sensitive to inhibition by phosphonoformic and phosphonoacetic acids than were those of other species studied so far. SDS/PAGE revealed that only a small number of brush-border membrane proteins were incorporated into the proteoliposomes. This reconstitution procedure should be useful for the purification and identification of the carrier protein.

Animals↗

PubMed1988

Effect of magnesium on ATP labelling by kidney brush border membrane.

1. Brush border membranes purified from rat kidney cortex were incubated in the presence of ATP and analysed by SDS polyacrylamide gel electrophoresis. 2. Quantitative analysis of phosphorylation was performed with a calibration curve obtained by autoradiography. 3. The presence of magnesium was required for the phosphorylation of membrane proteins. 4. EDTA completely inhibited the labelling of all bands, except for the alkaline phosphatase band. 5. In contrast, alkaline phosphatase was inhibited by 52, 65 and 85% in the presence of 1 mM bromotetramisole, 10 mM NaF and 10 mM Na arsenate respectively. 6. However these inhibitors had only minor effects on the labelling of other proteins. 7. High concentrations of magnesium caused a pronounced inhibition on the labelling of the alkaline phosphatase band but had no effect on the phosphorylation of other proteins.

Adenosine Triphosphate↗

PubMed1988

Outer membrane porin protein of Haemophilus influenzae type b: pore size and subunit structure.

The 40-kDa porin protein of Haemophilus influenzae type b was reconstituted into proteoliposomes. The relative rates of diffusion of small uncharged sugars across the channels formed by this protein were determined by measuring the rates of osmotic swelling of the liposomes. From these rates, a pore diameter of 1.8 nm was estimated using the Renkin equation. A chemical cross-linking technique was used to investigate the oligomeric structure of the 40-kDa porin. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis revealed the presence of porin dimers and trimers after reaction of the protein with dithio-bis-(succinimidyl propionate). These results confirmed that the porin of H. influenzae forms large water-filled channels and indicated that it probably exists as trimers in the outer membrane.

Bacterial Outer Membrane Proteins↗

PubMed1986

Properties of the porin of Haemophilus influenzae type b in planar lipid bilayer membranes.

The major outer membrane protein (40 kDa) of the bacterium Haemophilus influenzae type b is a porin which forms transmembrane permeability channels. It has an exclusion limit for oligosaccharides of about 1.4 kDa. When this protein was added to the aqueous phase which was bathing a planar lipid bilayer, it caused the conductance of the membrane to increase by several orders of magnitude. At low protein concentrations (2-10 pM), the conductance of the membrane increased in a stepwise fashion with an average single-channel conductance of 1.1 nS in 1 M KCl. Single-channel experiments were performed with a variety of different salts. The conductance of single channels was proportional to the specific conductance of the aqueous solution which was bathing the membrane. Current through the pores was proportional to the applied voltage, indicating that these pores are not voltage-controlled. The 40 kDa porin was very slightly cation-selective: the pores were about 1.6-times more permeable to potassium ions than to chloride ions. These properties of the 40 kDa porin are those of large water-filled channels and are characteristic of most bacterial porins. The single-channel conductance of the porin is, however, much smaller than might be expected from its exclusion limit. A model is proposed which could explain the differences in apparent pore size.

Bacterial Outer Membrane Proteins↗

PubMed1985

Transmembrane permeability channels across the outer membrane of Haemophilus influenzae type b.

Outer membranes of Haemophilus influenzae type b were fractionated to yield Triton X-100-insoluble material and lipopolysaccharide and phospholipids. Liposomes reconstituted from lipopolysaccharide and phospholipids were impermeable to sucrose (Mr, 342) and to a high-molecular-weight dextran (average Mr, 6,600). When the Triton X-100-insoluble material was introduced into the reconstituted liposomes, the vesicles became permeable to sucrose, raffinose (Mr, 504), and stachyose (Mr, 666) and fully retained dextrans of Mr greater than 1,500. Inulin (average Mr, 1,400) was tested for its efflux from the reconstituted outer membrane vesicles; 62% of the added inulin was trapped. The molecular weight exclusion limit for the outer membrane of H. influenzae type b was therefore estimated at approximately 1,400. A protein responsible for the transmembrane diffusion of solutes was purified from H. influenzae type b by extraction of whole cells with cetyl trimethyl ammonium bromide. When this extract was passed over DEAE-Sepharose, three protein-containing peaks (I, II, and III) were eluted. Peaks I and II contained mixtures of proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; when tested for their pore-forming properties, these proteins were unable to render liposomes of lipopolysaccharide and phospholipid permeable to sucrose. Peak III contained only one molecular species of protein of molecular weight 40,000; this protein acted as a porin in reconstituted vesicles. The molecular weight exclusion limit for 40,000-molecular-weight protein matched the estimate of approximately 1,400 which was determined for outer membranes. A series of homologous saccharides of increasing degree of polymerization was prepared from agarose by hydrolysis with beta-agarase and fractionation on gel filtration chromatography. These oligosaccharides of Mr, 936, 1,242, 1,548, and 1,854 were assayed for retention by the complete vesicles containing 40-kilodalton protein and lipopolysaccharide and phospholipids. All of these oligosaccharides were lost by efflux through the porin. Since the molecular conformation of the largest oligosaccharide is an elongated semirigid helix, it is suggested that the pore formed by the 40-kilodalton protein does not act as a barrier to the diffusion of this compound.

Amino Acids↗

PubMedDate not supplied

Fractionation of renal brush border membrane proteins with Triton X-114 phase partitioning.

Analysis of brush border membrane proteins by gel electrophoresis has revealed a complex polypeptide composition. We have investigated the use of Triton X-114 phase partitioning to fractionate such proteins on the basis of their degree of hydrophobicity. Each of the fractions was composed of a complex but distinct set of proteins. Most proteins were solubilized by Triton X-114 and partitioned into the detergent-poor fraction. Trehalase, gamma-glutamyl transpeptidase, and leucine aminopeptidase were well solubilized (greater than 80%) and enriched 5.1-, 3.9-, and 2.5-fold in the detergent-rich fraction. In contrast, alkaline phosphatase and 5'-nucleotidase were poorly solubilized. The specific activities of these enzymes were increased 2.7- and 2.3-fold in the insoluble protein fraction. Maltase was almost completely solubilized and partitioned into the detergent-poor fraction with a small enrichment factor (1.3). These results suggest that Triton X-114 phase partitioning could be useful as a first step in the purification of many brush border membrane proteins.

5'-Nucleotidase↗